Project Summary The ability of cancer cells to evolve and adapt to therapy is a challenge that limits treatment success and durability of responses. This is certainly the case in chronic lymphocytic leukemia (CLL), a malignancy of mature B cells that remains incurable, despite the potent cytolytic effects of both existing standard-of-care fludarabine-based combination chemotherapy, and newly developed targeted inhibitors such as venetoclax. To address this challenge, our active and ongoing CLL research program has focused on the search for consistent genetic loci associated with therapeutic resistance or progression through: (i) genetic characterization (by whole-exome sequencing analysis) of longitudinal samples collected from informative well- characterized clinical cohorts of patients that have relapsed following diverse CLL therapies; single cell transcriptome analysis of sequential samples across treatments and with progression from a subset of these patient cohorts; genome-wide knockdown and overexpression screens to systematically identify the underlying mechanistic basis of therapeutic resistance to venetoclax; and the generation of novel mouse models that are genetically faithful to human CLL, from which we can start to test novel drug combinations. As our studies have progressed, we have increasingly appreciated a need to integrate the genetic, expression and epigenetic information of drug-resistant cell populations so that we can gain a more principled approach to understanding this complex problem. To that end, the new innovative cell barcoding technology, COLBERT, developed by the Brock Lab, which provides the means to quantify, isolate, and analyze cell subpopulations, provides a highly informative approach to tackle the problem of understanding clonal evolution and therapeutic resistance in CLL. This technology platform utilizes integrated and expressed barcodes to track cell lineages and drive lineage-specific gene expression. Activation of reporter gene expression in specific subpopulations of interest enables isolation of purified cell lineages for downstream investigation in our genetic, epigenetic, and gene expression analysis workflows. We have already established the dose-ranges of drugs to test in the CLL cell line HG3, and have optimized workflows for introduction of lentivirus-based barcode libraries into the well- established MDR mouse line. Hence, we are well-poised to undertake the proposed collaborative work to comprehensively dissect resistance mechanisms underlying progression to cytotoxic chemotherapy and targeted BCL2 inhibitor in the context of cells harboring del(13q), the most common genetic alteration in CLL. Altogether, the proposed joint analyses serve to provide a fresh analytic framework for gaining vital information regarding the fitness, function and phenotype of distinct clones in the context of therapy, which we anticipate will be immensely beneficial to the design of the next generation of therapeutic approaches to overcome the evolutionary capacity of cancer.